Oral cancer, predominantly oral squamous cell carcinoma (OSCC), is a high impact disease in the oral cavity, affecting more than 34,000 people in the United States each year (American Cancer Society, 2007). Oral cancer is one of the cancers with the worst prognosis, with a 5-year survival rate of 40-50% (Greenlee, R. T. et al., CA Cancer J Clin, 50:7-33 (2000); Parkin, D. M. et al., CA Cancer J Clin, 55:74-108 (2005)). OSCC tumors arise through a series of molecular mutations that lead to uncontrolled cellular growth from hyperplasia to dysplasia to carcinoma in situ followed by invasive carcinoma. Major risk factors for OSCC include tobacco and alcohol consumption along with environmental and genetics factors (Brinkman, B. M. N. and Wong, D. T., Curr Opin Oncol, 18:228-233 (2006); Figuerido, M. L. et al., Drug Discov Today: Dis Mechan, 1:273-281 (2004); Hu, S. et al., Arthritis Rheum., 56:3588-600 (2007); Turhani, D. et al., Electrophor, 27:1417-1423 (2006)). OSCC is usually detected at late stages when the cancer has advanced and therefore results in poor prognosis and survival. Every individual has a unique prognosis due to the aggressiveness of their tumors therefore they do not behave similarly under the TNM staging system, which classifies tumors by size, lymph node metastasis and distant metastasis. Presently, surgery and radiotherapy are the primary treatments, but due to OSCC's location in the head and neck; this usually results in postoperative defects and functional impairments in patients (Thomson, P. J. and Wylie, J., Int J Oral Maxillofac Surg, 31:145-153 (2002)). Therefore, early disease detection is imperative because it can result in a more effective treatment with superior results.
Squamous cell carcinoma (SCC) of the oral cavity and oropharynx is the 6th most common cancer, with approximately 350,000 new cases worldwide annually. The overall 5-year survival rates for oral squamous cell carcinoma (OSCC) have remained low at approximately 30-40% for the past decades. Delayed detection is one of the main reasons for the high morbidity rate of oral cancer, suggesting an imperative need for developing biomarkers to improve early detection of oral cancers. Proteomic analysis of body fluids (e.g., saliva and serum) over the course of oral cancer progression holds promise to identify early detection biomarkers for human oral cancer.
Biomarkers are measurable biological and physiological parameters that can serve as indices for health-related assessments. Protein biomarkers are particularly powerful because they are amenable to simple blood or saliva tests and, once successfully developed, can benefit the cancer patients as simple clinical tools. In terms of identifying protein markers for cancer detection, a body fluid approach (e.g., saliva or blood) appears to be very attractive because it is easy to collect and process these body fluids as compared to tissue biopsies.
Saliva has gained notable attention as a diagnostic fluid because of its simple collection and processing, minimal invasiveness and low costs. Many researchers have studied salivary proteins as potential diagnostic markers for various diseases such as breast cancer, ovarian cancer, Sjögrens syndrome, hepatocellular carcinoma, leukoplakia and oral cancer (Ryu, O. H. et al., Rheumatol, 45:1077-1086 (2006); Streckfus, D. et al., Cancer Invest, 18:101-109 (2000); Rhodus, N. L. et al., Cancer Detect Prev, 29:42-45 (2005); Brailo, V. et al., Oral Oncol, 42(4):370-373 (2006); Yio, X. et al., Ann Clin Biochem, 29:519-522 (1992); Gorelik, E. et al., Cancer Epidemiol Biomarkers Prev, 14:981-987 (2005); Hu, S. et al., Arthritis Rheum., 56:3588-600 (2007)). These potential disease markers, if successfully developed, can lead to simple clinical tools for early detection and the monitoring of disease prognosis and treatment in saliva, a non-invasive body fluid (Kingsmore, S. F., Nat Rev Drug Discov, 5:310-321 (2006)).
Currently there are no reliable saliva biomarkers in the clinic for OSCC, however, some recent studies have suggested signature proteins in saliva from OSCC patients can be used for the disease detection. For instance, salivary proteins such as TNF-alpha, interleukin-1 (IL-1), IL-6, IL-8, CD44, fibronectin, defensin-1, cytokeratin 19 fragment (CYFRA 21-1), tissue polypeptide antigen, and cancer antigen CA125, were found over-expressed in OSCC patients (Mizukawa, N. et al., Oral Dis, 5(2):139-142 (1999); Franzmann, E. J. et al., Cancer Epidemiol Biomarkers Prev, 14(3):735-739 (2005); Rhodus, L. et al., Cancer Detect Prev, 29(1):42-45 (2005); Lyons, A. J. and Cui, N., J Oral Path Med, 29(6):267-270 (2000); Nagler, R. et al., Clin Cancer Res, 12(13):3979-3984 (2006); St. John, M. I. et al., Arch Otolaryngol Head Neck Surg, 130:929-935 (2004); Rhodus, N. L. et al., Cancer Detect Prev, 29(1):42-5 (2005); Brailo, V. et al., Oral Oncol, 42:370-373 (2006)). These proteins, if successfully validated in a large patient cohort, could be potentially useful for OSCC detection.
Analysis of the proteomic content in human saliva is important because it will not only contribute to understanding of oral health and disease pathogenesis but also form a foundation for the discovery of saliva protein biomarkers for human disease detection. Mass spectrometry (MS)-based proteomics has been successfully applied to identification of proteins and their PTMs in human whole and ductal saliva (Wilmarth, P. A. et al., J. Proteome Res., 3(5):1017-1023 (2004); Hu, S. et al., Proteomics, 5(6):1714-1728 (2005); Xie, H. et al., Mol. Cell. Proteomics, 4(11):1826-1830 (2005); Yates, J. R. et al., Anal. Chem., 78(2):493-500 (2006); Guo, T. et al., J. Proteome Res., 5(6):1469-1478 (2006); Hu, S. et al., Ann. N.Y. Acad. Sci, 1098:323-329 (2007)). Many of these studies were performed using shotgun proteomics, which is based on multidimensional separation, tandem MS (MS/MS) and database searching algorithms. Shotgun proteome analysis is very efficient in cataloguing and profiling of proteins, whereas 2-D gel electrophoresis coupled with MS (2-DE/MS) allows mapping out the proteome at protein level and visualization of protein modifications and isoforms (Hirtz, C. et al., Proteomics, 5(17):4597-4607 (2005); Walz, A. et al., Proteomics, 6(5):1631-1639 (2006)).
Profiling of salivary glycoproteins and proteins in distinct families has been demonstrated lately. The selective enrichment of glycoproteins followed by liquid chromatography-tandem MS (LC-MS/MS) profiling may appear to be a promising approach for finding biomarker and therapeutic targets in cancers (Ramachandran, P. et al., J. Proteome Res., 5(6):1493-1503 (2006)). Analysis and characterization of cystatins, histatins, proline-rich proteins and their fragments in saliva provides further insight in assessment of their functions in the oral cavity (Inzitari, R. et al., Proteomics, 6(23):6370-6379 (2006); Inzitari, R. et al., Proteomics, 5(3):805-815 (2005); Messana, I. et al., J. Proteome Res., 3(4):792-800 (2004); Castagnola, M. et al., J. Biol. Chem., 279(40):41436-41443 (2004); Lupi, A. et al., Proteomics, 3(4):461-467 (2003)). In addition, a salivary proteome database (http://www.hspp.ucla.edu) has been established to centralize the acquired proteomic data and annotate the identified saliva proteins. These databases are fully accessible to the public for query of the identified proteins, which are linked to public protein databases. With the data deposited and centralized, the processes of integrating large-scale datasets from a variety of laboratories and conducting comparative analysis of saliva proteome to other body fluid proteomes can now begin.
Early diagnosis of oral cancers is imperative, as successful treatment of these cancers often depends on early detection. Considering that approximately 10% of the general population have oral mucosal abnormalities, and that precancerous and early cancerous lesions rarely demonstrate distinct clinical characteristics, there is a growing realization that some premalignant and early cancerous lesions are not readily detectable by visual inspection. Therefore, the integration of early detection and screening based on protein biomarkers, in conjunction with a conventional oral examination, is extremely important. This clearly requires comparative proteome analysis of oral pre-cancer and cancer samples in order to achieve protein markers for truly early detection of OSCC.
The present invention fulfills a need in the art for both salivary oral cancer protein biomarkers and practical methods of detecting these saliva-based biomarkers. The present invention provides saliva-based diagnostic biomarkers of oral squamous cell carcinoma (OSCC) and periodontal disease. The present invention also provides methods of diagnosing and distinguishing both periodontal disease and OSCC.